[Show abstract][Hide abstract] ABSTRACT: p53 is a crucial tumor suppressor that is mutated or deleted in a majority of cancers. Exactly how p53 prevents tumor progression has proved elusive for many years; however, this information is crucial to define targets for chemotherapeutic development that can effectively restore p53 function. Bioactive sphingolipids have recently emerged as important regulators of proliferative, apoptotic and senescent cellular processes. In this study, we demonstrate that the enzyme sphingosine kinase 1 (SK1), a critical enzyme in the regulation of the key bioactive sphingolipids ceramide, sphingosine and sphingosine-1-phosphate (S1P), serves as a key downstream target for p53 action. Our results show that SK1 is proteolysed in response to genotoxic stress in a p53-dependent manner. p53 null mice display elevation of SK1 levels and a tumor-promoting dysregulation of bioactive sphingolipids in which the anti-growth sphingolipid ceramide is decreased and the pro-growth sphingolipid S1P is increased. Importantly, deletion of SK1 in p53 null mice completely abrogated thymic lymphomas in these mice and prolonged their life span by ~30%. Deletion of SK1 also significantly attenuated the formation of other cancers in p53 heterozygote mice. The mechanism of p53 tumor suppression by loss of SK1 is mediated by elevations of sphingosine and ceramide, which in turn were accompanied by increased expression of cell cycle inhibitors and tumor cell senescence. Thus, targeting SK1 may restore sphingolipid homeostasis in p53-dependent tumors and provide insights into novel therapeutic approaches to cancer.
[Show abstract][Hide abstract] ABSTRACT: Ceramidases (CDase) are enzymes that catalyze the hydrolysis of N-acyl linkage of ceramide (Cer) to generate sphingosine and free fatty acids. In this study we report the purification and characterization of a novel second type of neutral ceramidase from rat brain (RBCDase II). Triton X-100 protein extract from rat brain membrane was purified sequentially using Q-Sepharose, HiLoad16/60 Superdex 200pg, heparin-Sepharose, phenyl-Sepharose HP, and Mono Q columns. After Mono Q, the specific activity of the enzyme increased by ~15,000-fold over that of the rat brain homogenate. This enzyme has pH optima of 7.5, and it has a larger apparent molecular weight (110kDa) than the previously purified (90kDa) and characterized neutral rat brain CDase (RBCDase I). De-glycosylation experiments show that the differences in molecular mass of RBCDase I and II on SDS-PAGE are not due to the heterogeneity with N-glycan. RBCDase II is partially stimulated by Ca(2+) and is inhibited by pyrimidine mono nucleotides such as TMP and UMP. This finding is significant as it demonstrates for the first time an effect by nucleotides on a CDase activity. The enzyme was also inhibited by both oxidized and reduced GSH. The effects of metal ions were examined, and we found that the enzyme is very sensitive to Hg(2+) and Fe(3+), while it is not affected by Mn(2+). EDTA was somewhat inhibitory at a 20mM concentration.
Biochimica et Biophysica Acta 01/2011; 1811(4):242-52. · 4.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Objectives: The objective of this study was to assess the therapeutic role of a C18-ceramide analogue, C18-pyridinium ceramide, in autophagy induced cell death in HNSCC tumors.
Methods: This study utilized in situ induction of CerS1/C18-ceramide using tetracycline inducible stable cell lines. Moreover, luciferase expressing flank and orthotopic (HNSCC) tumor models were utilized to assess the therapeutic potential of C18-pyridinium ceramide. Furthermore, western blot analysis, siRNA, quantitative PCR, and confocal microscopy were utilized. Atg5 WT and -/- cell lines were used to assess the role of Atg5 in autophagy induction.
Results: In situ induction of CerS1/C18-ceramide using tetracycline inducible stable cell lines resulted in a significant suppression of tumor growth. Interestingly, cell survivial studies show C18-pyridinium ceramide (LCL-461) results in either growth inhibition or cell death in UMSCC-1, UMSCC-22A, and WT Atg5 MEFs. Mechanistically, growth inhibition is a result of autophagy induction since these treatments induced lipidation of LC3 (light chain 3/Atg8), a well-known marker of autophagy. Autophagy related genes (Atg), specifically Atg5, have been shown to be vital to the molecular mechanism autophagosome maturation. Interestingly, LCL-461 treatment of Atg5 WT MEFs caused LC3 lipidation but the Atg5 -/- cells did not. Noticeably, WT Atg5 but not Atg5 -/- MEFs resulted in significant growth inhibition when treated with LCL-461.
Conclusion: In conclusion, these results suggest that the ceramide-18 analogue, LCL-461, may interact with the Atg5 protein inducing autophagosome formation resulting in growth inhibition and cell death of HNSCC tumors.
Support: This work was supported by the National Institutes of Health, National Research Service Award Institutional Research Training Grant (T32) MUSC CDM, CA-97132 to YAH, DE-01657 to BO
[Show abstract][Hide abstract] ABSTRACT: Sphingomyelin hydrolysis in response to stress-inducing agents, and subsequent ceramide generation, are implicated in various cellular responses, including apoptosis, inflammation and proliferation, depending on the nature of the different acidic or neutral sphingomyelinases. This study was carried out to investigate whether the neutral Mg(2+)-dependent neutral sphingomyelinase-2 (nSMase2) plays a role in the cellular signaling evoked by TNFalpha and oxidized LDLs, two stress-inducing agents, which are mitogenic at low concentrations and proapoptotic at higher concentrations.
For this purpose, we used nSMase2-deficient cells from homozygous fro/fro (fragilitas ossium) mice and nSMase2-deficient cells reconstituted with a V5-tagged nSMase2. We report that the genetic defect of nSMase2 (in fibroblasts from fro/fro mice) does not alter the TNFalpha and oxidized LDLs-mediated apoptotic response. Likewise, the hepatic toxicity of TNFalpha is similar in wild type and fro mice, thus is independent of nSMase2 activation. In contrast, the mitogenic response elicited by low concentrations of TNFalpha and oxidized LDLs (but not fetal calf serum) requires nSMase2 activation.
nSMase2 activation is not involved in apoptosis mediated by TNFalpha and oxidized LDLs in murine fibroblasts, and in the hepatotoxicity of TNFalpha in mice, but is required for the mitogenic response to stress-inducing agents.
PLoS ONE 01/2010; 5(3):e9826. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have utilized several peptide specific antisera directed against the C-terminals (Wetsel et al, 1992) of several protein kinase C (PKC) isozymes (α, β1, β11, γ, δ, ϵ, ξ) to delineate the cellular localization of these PKC isozymes in rat retina. Antisera against PKC β1, β11, γ, δ and ϵ were non-reactive in frozen rat retina sections, whereas, anti PKCα was strongly reactive with the outer plexiform, inner plexiform and nerve fiber cell layers. The most specific localization of immunoreactivity was observed with PKCξ, which reacted strongly and exclusively with photoreceptor inner segments, but not outer segments. Immunoblot analysis of whole rat retina homogenate showed that anti-PKCα recognized an antigen of 80kD and anti-PKCξ recognized a 72kD protein. Immunolocalization of PKCξ to photoreceptor inner segments and possible functional significance are discussed.
[Show abstract][Hide abstract] ABSTRACT: The sphingolipid ceramide is intimately involved in the growth, differentiation, senescence, and death of normal and cancerous cells. Mitochondria are increasingly appreciated to play a key role in ceramide-induced cell death. Recent work showed the C16-pyridinium ceramide analogue LCL-30 to induce cell death in vitro by mitochondrial targeting. The aim of the current study was to translate these results to an in vivo model. We found that LCL-30 accumulated in mitochondria in the murine colorectal cancer cell line CT-26 and reduced cellular ATP content, leading to dose- and time-dependent cytotoxicity. Although the mitochondrial levels of sphingosine-1-phosphate (S1P) became elevated, transcription levels of ceramide-metabolising enzymes were not affected. In mice, LCL-30 was rapidly absorbed from the peritoneal cavity and cleared from the circulation within 24 h, but local peritoneal toxicity was dose-limiting. In a model of subcutaneous tumour inoculation, LCL-30 significantly reduced the proliferative activity and the growth rate of established tumours. Sphingolipid profiles in tumour tissue also showed increased levels of S1P. In summary, we present the first in vivo application of a long-chain pyridinium ceramide for the treatment of experimental metastatic colorectal cancer, together with its pharmacokinetic parameters. LCL-30 was an efficacious and safe agent. Future studies should identify an improved application route and effective partners for combination treatment.
British Journal of Cancer 02/2008; 98(1):98-105. · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.
[Show abstract][Hide abstract] ABSTRACT: The degradation of biogenic amines by monoamine oxidase A (MAO-A) generates reactive oxygen species (ROS) which participate in serotonin and tyramine signaling. This study aimed to investigate the role of ROS in the mitogenic signaling activated during tyramine and serotonin oxidation by MAO-A in smooth muscle cells (SMC). Incubation of SMC with serotonin or tyramine induced intracellular ROS generation, and a signaling cascade involving metalloproteases and the neutral sphingomyelinase-2 (nSMase2, the initial step of the sphingolipid pathway), ERK1/2 phosphorylation, and DNA synthesis. Silencing MAO-A by siRNA, pharmacological MAO-A inhibitors (pargyline and Ro41-1049), and the antioxidant/ROS scavenger butylated hydroxytoluene (BHT) inhibited the signaling cascade, suggesting that ROS generated during tyramine oxidation by MAO-A are required. The MMP inhibitor Batimastat, MMP2-specific siRNA, and MMP2 deletion (MMP2(-/-) fibroblasts) blocked nSMase activation and SMC proliferation, suggesting a role for MMP2 in this signaling pathway. Silencing nSMase2 by siRNA did not inhibit ROS generation and MMP2 activation, but blocked SMC proliferation induced by tyramine, suggesting that nSMase2 is downstream MMP2. These findings demonstrate that H(2)O(2)-generated during tyramine oxidation by MAO-A triggers a stress-induced mitogenic signaling via the MMP2/sphingolipid pathway, which could participate in excessive remodeling and alteration of the vascular wall.
Free Radical Biology and Medicine 08/2007; 43(1):80-9. · 5.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Over the last two decades, sphingolipids have emerged as important cell regulators. Included in this lipid class are the lipid mediator sphingosine, its phosphorylated derivative sphingosine-1-phosphate, and ceramide. In humans, the major known functions of sphingolipid-mediated cell regulation is the modulation of signaling pathways which control fundamental cellular processes including cell division, senescence, apoptosis, angiogenesis, and differentiation [40, 41, 74], all of direct relevance to cancer pathogenesis and progression. Because of these myriad activities, the enzymes that generate sphingolipid mediators are potential targets for cancer treatment. Thus, characterization of these enzymes with respect to activity, regulation, localization, and cellular function is fundamental to developing strategies for modulating sphingolipid levels in vivo. The yeast system has emerged as an invaluable tool for the identification, cloning, and characterization of enzymes of sphingolipid metabolism. Furthermore, insights gained from yeast studies of sphingolipid metabolism and function continue to push the forefront of sphingolipid research, especially since the roles of sphingolipids in stress and other cell responses appear to be conserved from yeast to human.
[Show abstract][Hide abstract] ABSTRACT: The protein kinase C (PKC) family of isoenzymes has been shown to regulate a variety of cellular processes, including receptor desensitization and internalization, and this has sparked interest in further delineation of the roles of specific isoforms of PKC in membrane trafficking and endocytosis. Recent studies have identified a novel translocation of PKC to a juxtanuclear compartment, the pericentrion, which is distinct from the Golgi complex but epicentered on the centrosome. Sustained activation of PKC (longer than 30 min) also results in sequestration of plasma membrane lipids and proteins to the same compartment, demonstrating a global effect on endocytic trafficking. This review summarizes these studies, particularly focusing on the characterization of the pericentrion as a distinct PKC-dependent subset of recycling endosomes. We also discuss emerging insights into a role for PKC as a central hub in regulating vesicular transport pathways throughout the cell, with implications for a wide range of pathobiologic processes, e.g. diabetes and abnormal neurotransmission or receptor desensitization.
Cellular and Molecular Life Sciences CMLS 03/2007; 64(3):263-70. · 5.62 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: As of January 2005, there were 1020 gene therapy clinical trials ongoing worldwide with 675 or 66.2% devoted to cancer gene therapy. The majority are occurring in the US and Europe (http://www.wiley.co.uk/genetherapy/clinical/). At the present time, to our knowledge there are no trials that employ gene delivery of Fas Ligand (FasL). As an important note, and in contrast to somatic cell therapy trials, there are no reported deaths due to therapeutic vector administration in any cancer gene therapy trial. That said, from our studies and from the published literature, the issue of gene delivery remains the major obstacle to successfully employing gene therapy for cancer treatment. Numerous laboratories are studying this with many different approaches. My co-workers and I have focused on the delivery issue by using various approaches that address tumor targeting and transgene expression. In addition, we are focusing on enhancing tumor cell killing via the bystander effect and through use of small molecules to enhance bystander activity.
Cancer Gene Therapy 01/2007; 13(12):1045-51. · 2.95 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Sphingolipids are signaling molecules in a range of biological processes. While sphingosine-1-phosphate (S1P) is thought to be abundantly stored in platelets and released upon stimulation, knowledge about the distribution and function of other sphingolipids in blood is lacking.
To analyze the sphingolipid content of blood components with special emphasis on dynamic changes in platelets.
Blood components from mice and humans were prepared by gradient centrifugation and analyzed by liquid chromatography-mass spectrometry. Additionally, murine platelets were activated in vitro and in vivo.
Isolated non-activated platelets of mice were devoid of S1P, but instead contained dihydrosphingosine-1-phosphate (dhS1P), along with a high concentration of ceramide. Activation of platelets in vitro led to a loss of dhS1P and an increase in sphingosine, accompanied by a reduction of ceramide content. Platelet activation in vivo led to an immediate and continuous rise of dhS1P in plasma, while S1P remained stable. The sphingolipid distribution of human blood was markedly different from mice. Human platelets contained dhS1P in addition to S1P.
Mouse platelets contain dhS1P instead of S1P. Platelet activation causes loss of dhS1P and breakdown of ceramide, implying ceramidase activation. Release of dhS1P from activated platelets might be a novel signaling pathway. Finally, the sphingolipid composition of mouse and human blood shows large differences, which must be considered when studying sphingolipid biology.
Journal of Thrombosis and Haemostasis 01/2007; 4(12):2704-9. · 6.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Despite local and systemic therapies, the National Cancer Institute estimates that prostate cancer will cause over 30,000 deaths in 2006. This suggests that additional therapeutic approaches are needed. The chicken anemia viral protein Apoptin causes tumor-selective apoptosis in human tumor lines independent of p53 and Bcl-2 status. Tet-regulated expression of Apoptin from an adenoviral vector showed cytotoxicity in DU145, PC-3, and LNCaP tumor cells regardless of expression of p53, Bcl-2, Bcl-xL, Bax, survivin, FLIP(S), XIAP, or CIAP. Apoptin expression caused an increase in the tumor suppressor lipid ceramide, which regulates the cellular stress response. Interestingly, 10 of 15 primary prostate cancers examined by Western blotting overexpressed acid ceramidase (AC), suggesting that ceramide deacylation might serve to negate elevated levels of ceramide, creating a more antiapoptotic phenotype. This was confirmed in AC-overexpressing cells in which we observed decreased sensitivity to apoptosis following treatment with Apoptin. Addition of the AC inhibitor LCL204, in combination with Apoptin, augmented cell killing. This effect was also demonstrated in vivo in that Apoptin and LCL204 cotreatment significantly reduced tumor growth in DU145 xenografts (P<0.05). Taken together, our data demonstrated that Apoptin is a promising therapeutic agent for prostate cancer and that its function is improved when combined with acid ceramidase inhibitors.
[Show abstract][Hide abstract] ABSTRACT: Emerging information on sphingolipid metabolism and signaling is leading to a better understanding of cellular processes such as apoptosis, cancer, cell survival and aging. In this review, we discuss the involvement of sphingolipids in these processes and focus on underlying mechanisms based on sphingolipid:protein interactions. Due to the inherent difficulty of studying lipids, we discuss techniques that are useful in the elucidation of these interactions. We classify sphingolipid-binding proteins into four main classes: receptor, effector, enzyme, and transporter. Known structures of sphingolipid-binding proteins are surveyed, and sphingolipid-binding characteristics are described, acknowledging the limitations that there are presently insufficient protein:sphingolipid complexes for more definitive conclusions on this topic. Finally we summarize relevant literature to better inform the reader about sphingolipid:protein interactions.
Biochimica et Biophysica Acta 09/2006; 1761(8):927-46. · 4.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: INTRODUCTION AND OBJECTIVE: RCC is the sixth leading cause of cancer death in USA. One third of patients present with metastatic disease. Current systemic therapies have limited results. Gene therapy including pro-apoptotic gene therapy is a promising tool under investigation for treatment of RCC. However, inability to deliver lethal genes to all tumor cells is a problem. FasL is a pro-apoptotic gene that is a member of the Tumor Necrosis Factor family. Although ACHN cells were resistant to convention Fas monoclonal Ab (CH-11), we were able to induce apoptosis by AdGFPFasLTET gene therapy. Ceramide generation was observed after treatment. Ceramide, the basic unit of sphingolipid, is involved in cell cycle arrest and/ or apoptosis. On Using Ceramide or ceramide analogue, we were able to sensitize ACHN cells to CH-11. This indicates that ceramide plays a significant role in sensitizing ACHN to gene therapy.METHODS: ACHN treated with CH-11, AdGFPFasLTETTET, or AdGFP. AdGFPFasLTETTET is replication-deficient adenovirus containing a modified murine Fas ligand gene fused to green fluorescent protein (GFP), with a promoter can down-regulate gene expression in presence of doxycycline. AdGFP that contain the GFP gene alone was used as a control. Viability of the cells was assessed using MTS assay. Cells were also evaluated for apoptosis by alteration of mitochondrial membrane potential (MMP) by FACS and caspase 3/7 assay. Ceramide level with AdGFPFasLTET treatment was measured by HPLC-Ms in Lipidomics core. C6-De erythro-ceramide and lysosomotropic ceramide analogue LCL-385 were combined with CH-11 and viability was assessed by MTS. Apoptosis was confirmed using casapase 3/7 assay.RESULTS: ACHN cells were resistant to apoptosis when treated with CH-11. However, ACHN were extremely sensitive to AdGFPFasLTET while minimal cytotoxicity was seen with control virus AdGFP at the same MOI. Caspase 3/7 was increased by treatment with AdGFPFasLTET and not with AdGFP or CH-11. Increased ceramide level was observed with treatment with AdGFPFasLTET. C6-De-Erythro ceramide or LCL-385 were able to sensitize ACHN cells to CH-11 as assessed by MTS and casapse 3/7 assay.CONCLUSIONS: ACHN cells are extremely sensitive to AdGFPFasLTET treatment and resistant to CH-11. AdGFPFasLTET treatment results in Ceramide generation. Treating ACHN with Ceramide or ceramide analogue lead to sensitizing these cells to CH-11. These findings support the significance to investigate further role of ceramide in sensitizing resistant cancer cells to gene therapy or other chemotherapy.
[Show abstract][Hide abstract] ABSTRACT: Introduction and Objectives: Current treatment for advanced PCa is mainly palliative. Recurrence after androgen ablation with more progressive disease typically occurs within 18 months of treatment. Gene therapy is an alternative therapy currently under investigation and is hampered by inefficient delivery to all cancer cells. Sensitizing cancer cells to gene therapy (GT) may augment its effect and increases the bystander effect. FasL mediates apoptosis through caspases and survivin is a downstream inhibitor. We have previously shown that FasL GT can induce apoptosis in prostate cancer cells and can generate a bystander effect. Furthermore, it is becoming increasingly evident that sphingolipids in general and ceramide in particular play a role in prostate cancer regulation. LCL-204, Ceramide analogue, is able to initiate apoptosis through lysosomes. Herein, we show that LCL-204 can sensitize DU145 PCa cells to FasL GT and that survivin may play an important role in this effect.Methods: MTS assay to asses cell viability. Lysotracker red to demonstrate Lysosomal pH changes and methyl dancyl cadaverine dye to show autophagic vacuoles. Proteasomal activity assessed by proteasomal assay. Western blot to show protein changes. Transfection and siRNA to show effect of survivin.Results: pretreatment with low doses of LCL-204 sensitized DU145 cells to AdGFPFasLTET . LCL-204 caused Lysosomal pH changes within few minutes after treatment as demonstrated by FACS and increased autophagic vacuoles. This is followed by increased proteasomal activity and down-regulation of anti-apoptotic protein survivin. We demonstrated increased proteasomal activity was partial responsible for survivin degradation. This was partially inhibited by proteasome inhibitors (lactacystin and MG132) as demonstrated by western blot. We also protected DU145 cells against apoptosis by AdGFPFasLTET by Transfecting DU145 cells with survivin-GFP DNA. Finally, we were able to sensitize DU145 cells to FasL induced apoptosis by Targeting survivin using siRNA.Conclusion: Survivin plays a role in protecting cells again Fas induced apoptosis. Down regulation of survivin using LCL-204 can augment the effect of Fas induced apoptosis. LCL-204 can be a potential drug used to augment the effect of AdGFPFasLTET in prostate cancer.
[Show abstract][Hide abstract] ABSTRACT: In addition to their crucial role in membrane structure in Saccharomyces cerevisiae, sphingolipids serve vital roles in various aspects of yeast biology including endocytosis, intracellular protein transport and stress responses. Although previous studies have unequivocally demonstrated the sphingolipid requirements for these processes, few studies have contributed mechanistic information. We have used a systems approach including microarray, lipidomics and metabolic modelling to better understand (i) biochemical relationships between various branches of sphingolipid metabolism and pathways and contributing pathways such as fatty acid metabolism and phospholipid synthesis, (ii) the changes in cellular sphingolipid composition under various conditions and (iii) the effects of these changes on the transcriptional profiles and subsequently, cell phenotypes. Thus far, these approaches have indicated roles for sphingolipids in major transcriptional changes in response to heat stress, carbon source utilization, sporulation, cell wall integrity and other basic cellular functions. Although the yeast genome is fully sequenced, nearly 50% of all transcribed open reading frames remain uncharacterized with regard to cellular function; therefore, a major advantage of this approach is the ability to identify both biochemical and biological roles for enzymes and their products within broad cellular contexts.
Biochemical Society Transactions 12/2005; 33(Pt 5):1166-9. · 2.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Diacylglycerol (DAG) was discovered as a potent lipid second messenger with protein kinase C (PKC) as its major cellular target more than 25 years ago. There is increasing evidence of significant complexity within lipid signaling, and the classical DAG-PKC model no longer stands alone but is part of a larger bioactive lipid universe involving glycerolipids and sphingolipids. Multiple layers of regulation exist among PKC- and DAG-metabolizing enzymes such as phosphatidylcholine (PC)-specific phospholipase D, and cross-talk exists between the glycerolipid and sphingolipid pathways, with PKC at the center. Currently, there is intense interest in the question of whether DAG derived from PC can function as a lipid second messenger and regulate PKC analogous to DAG derived from phosphatidylinositol-4,5-bisphosphate (PIP2). To address these issues and incorporate DAG-PKC and other signaling pathways into an expanded view of cell biology, it will be necessary to go beyond the classical approaches and concepts.
Cellular and Molecular Life Sciences CMLS 08/2005; 62(13):1448-61. · 5.62 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Prostate cancer (PCa) is the most prevalent cancerin men and the second leading cancer death.Nearly 50% of patients have recurrence after definitive local therapy indicating an increased need for more effective therapies. We previously demonstrated (Hyer ML et al. Mol Ther 2(4):348–358, 2000) that adenovirus (AdGFPFasLTET) mediated overexpression of a GFPFasL fusion protein overcomes Fas resistance in DU145 cells induicng apoptosis. Bystander activity is also produced due to FasL presentation on apoptotic vesicles. However, some fraction of cancers, i.e DU145 xenografts, are resistant to bystander-mediated apoptosis (Hyer ML et al. Can Gene Ther10(4):330–339, 2003). We can promote sensitization to FasL bystander induced apoptosis by modification of ceramide metabolism.Ceramide is a sphingolipid involved in regulation of many cellular processes, as differentiation, growth suppression, and apoptosis and called a tumor suppressor lipid. Elevated ceramide generates a proapoptotic phenotype. Acid ceramidase (AC), upregulated in 42% of PCa, decreases ceramide by deacylation generating sphingosine and a free fatty acid. Sphingosine, is phosphorylated by sphingosine kinase yielding sphingosine 1-phosphate, promotes cell migration and angiogenesis.Here, we use a ceramide analogue, AC inhibitor (LCL204), that has lysomotropic characteristics to augment AdGFPFasLTET therapy in DU145 cells by several folds. We believe LCL204 acts through several different mechanisms to enhance FasL induced apoptosis. First, LCL204 targets the lysosome resulting in lysosomal destabilization as demonstrated by FACS analysis using LysoTracker Red, and by translocation of lysosomal cathepsins (B/D)to the cytosol. LCL204 also induces proteolytic degradation of AC, resulting in sphingosine peak followed by ceramide elevation within the first hour. The lysosomal and ceramide effects are proximal to increased proteasome activity and down-regulation of antiapoptotic proteins. We also have evidence that LCL204 destabilizes the mitochondrial membrane followed by cytosolic relocation of cytochrome c and caspase 9 activation. Concomitantly, we demonstrate that LCL204 downregulates sphingosine kinase activity as assayed enzymatically. These events collectively push the cancer cells towards a proapoptotic phenotype. When administered alone, AdGFPFasLTET induces biphasic ceramide accumulation with a primary peak at 6 hours followed by a secondary peak at 18 hours that remains elevated until apoptosis. Finally, the combination of AdGFPFasLTET and LCL204 invivo is able to reduce tumor growth in nude mice more effectively than either treatment alone.To summarize, AC inhibitor elevates intracellular ceramide, and sensitizes DU145 cells to FasL-induced apoptosis by at least two mechanisms; increasing intracellular ceramide, and by downregulation of antiapoptotic proteins. Finally, LCL204 is useful as a single or in combination with FasL gene therapy for treatment of hormone refractory prostate cancer xenografts.
[Show abstract][Hide abstract] ABSTRACT: Sphingolipids, historically described as potential reservoirs for bioactive lipids, presently define a new family of cellular mediators, joining the well-established glycerolipid-derived mediators of signal transduction such as diacylglycerol, phosphatidylinositides, and eicosanoids. Sphingolipid metabolism is clearly involved in the regulation of cell growth, differentiation, and programmed cell death. Indeed, a majority of the greater than four thousand studies conducted on sphingolipids during the past five years were investigations of the role of sphingolipids as cellular bioregulators. Studies spanning more than a decade have shown multiple interactions and intersections of the sphingolipid-mediated pathways and the eicosanoid pathway. This review will discuss the emerging mechanisms by which sphingolipids induce inflammatory responses via the eicosanoid pathway in addition to linking previous literature on sphingolipids and inflammation with newer findings of distinct roles for sphingosine-1-phosphate in regulating cyclooygenase-2 and ceramide-1-phosphate in the regulation of cytosolic phospholipase A2alpha. Finally, the relationship between bioactive sphingolipids and inflammation is discussed.
Current Molecular Medicine 07/2004; 4(4):405-18. · 4.20 Impact Factor